The invention relates to the operation and embodiment of a time-of-flight mass spectrometer for acquiring spectra of either primary or daughter ions with high mass precision.
In biochemistry, it is not only the saving of time and money that makes it desirable to achieve a high analysis throughput: in many application fields, the instability of the samples makes it essential that analytic procedures are carried out rapidly. Whereas in combinatorial chemistry the saving in time when analyzing tens of thousands of samples may be the most significant factor, in proteomics it must be considered that the proteins of a proteome, following their (for example, gel-electrophoretic) separation, purification and other sample preparation processes, are susceptible to oxidative, thermal or other types of decomposition, since they are no longer protected by their former association with other proteins and by the environment of a biological solution. This means that the thousands of proteins constituting a proteome should be analyzed within 24 hours, if possible, and at most 48 hours following their separation.
It is thus not only desirable but essential to achieve a high sample throughput for biochemical analysis.
Nowadays mass spectrometers are used for many biochemical analyses, and in particular for protein analysis. Most of these are time-of-flight mass spectrometers, in which the samples are ionized by laser desorption. Although modem mass spectrometers of this type are fitted with sample inlet systems which permit a large number of samples (384, 764 or even 1536 samples, for instance) to be placed on the sample supports, diverse problems associated with the fast analysis of these samples still remain, and these problems hinder high analysis throughput. These problems include both technical difficulties associated with the mass spectrometers being used and with the procedures employed, as well as difficulties with the reproducible preparation of the samples for ionization.
In proteome research the highest priority is to identify the individual proteins as rapidly as possible, but then also to identify differences from proteins that are already known. The identification is usually achieved by measurement of the precise masses of the peptides generated by enzymatic (preferably tryptic) digestion. The mixture of digestion peptides is subjected to MALDI analysis and a so-called xe2x80x9cfingerprint spectrumxe2x80x9d is generated. A special search algorithm is then used to compare the list of precise masses measured with the contents of a protein sequence database, frequently already yielding definite identifications. If, however, uncertainties result from ambiguity, or from masses that do not precisely match, then the peptides in question are investigated using a daughter ion analysis, and as a rule this will provide unambiguous answers.
In the type of time-of-flight mass spectrometry most often used here, ions of an analyte substance are created in an ion source by means of a short laser pulse. The ions are accelerated to a high energy in a short acceleration path, sent through a field-free flight-section, and measured by a time-resolving ion detector. Since all the ions have the same energy, the flight time of the ions measured in this way permits the determination of the mass, m, of the ions, or, more precisely, their mass to charge ratio, m/z.
Note: for the sake of simplicity, in the following reference will only be made to the mass, m, even though in mass spectrometry always the mass to charge ratio, m/z, is measured, where z is the number of elementary charges carried by the ion. Since many methods of ionization, such as, for instance, the matrix assisted laser desorption and ionization (MALDI) that is preferably used here, predominantly produce ions with only a single charge (z=1), this distinction is of little practical relevance here.
Since a single MALDI process only generates relatively few ions, the mass spectrometric analysis of a sample based on MALDI requires the summation of between 50 and 200 individual spectra in order to obtain a useful sum spectrum. In other words, between 50 and 200 laser pulses must be separately applied, each generating ions which are independently measured as an individual spectrum for inclusion in the sum spectrum. The problems mentioned above now have three principal aspects:
Up to now, the complex sequence of voltage pulses described below, which must be triggered each by a laser pulse, is simply switched on for acquiring the spectra and switched off in order to prepare for the next sample analysis. The electrical and thermal equilibria will never really balance as a result. Only under painfully maintained equilibrium conditions, however, is it possible to accurately reproduce all the voltage pulses, and this in turn is critical for the quality of the spectra.
To acquire the spectra of daughter ions, it is at present necessary to readjust the ion source potentials between one sample and the next, and within one sample even for the several daughter ion spectra from different precursor ions, in order to achieve optimal mass resolution for the precursor ion selection. Each adjustment, however, again disturbs the equilibrium of the electronics.
The preparation of the samples on the sample supports must be so uniform, and so homogenous throughout the samples, that the process of the quasi-explosive evaporation and ionization of the samples by the laser pulse is entirely reproducible, so that the 50 to 200 individual spectra all have the same quality, and that there are no variations in the flight times. This is hard to achieve.
An almost obvious solution for the first problem would be to allow the sequence of voltage pulses to run periodically at some fundamental frequency. The sequence of pulses in the ion source (and in turn all the other sequences of pulses), however, is triggered by the laser pulse itself, in order to eliminate the relatively dramatic effects of the laser""s slightly irregular ignition delay. For most lasers it is, on the other hand, inappropriate simply to allow the laser to operate continuously at a high pulse rate merely for the purpose of keeping the electronics in equilibrium. Not only might the samples on the sample support be damaged by the laser irradiation, but the laser itself only has a limited life time. The life time of the laser would be considerably reduced by such continuous operation. Thus the number of laser shots within the life time must be carefully budgeted, particularly when high sample throughput procedure is aimed for.
The ions generated by laser desorption frequently possess initial velocities that are not the same for all the ions. In order to achieve a high mass resolving power, velocity focusing by a Mamyrin ion reflector has become widely used, followed by a second field-free flight path. The ion reflector usually has two stages. In the first stage the ions are decelerated strongly, but in the second stage only gently. Faster ions penetrate further into the relatively weak linear deceleration field in the second stage of the reflector than do the slower ions, and therefore cover a greater distance. If the two deceleration fields have the correct relationship, this longer pathway compensates precisely for the higher flight speed, resulting in an increased mass resolution.
One of the most commonly used ion sources makes use of matrix assisted laser desorption and ionization (MALDI). The analyte molecules are embedded in a matrix substance, on a sample support plate. A pulse of laser light between 1 and 5 nanoseconds in length creates a cloud of molecules of both the matrix and analyte substance. The cloud expands adiabatically into the surrounding vacuum, giving the molecules in the cloud a greater spread of velocities. In this cloud, analyte molecules are continuously ionized by transfer of protons from the matrix ions, so that the analyte ions not only show a spread of velocities, their formation times are also spread.
A reflector is not able to focus this simultaneous spread of both speed and creation time. For this reason, a further method for improvement of mass resolution has been widely adopted for MALDI, comprising a delay in the acceleration. The basic principle behind the improvement in mass resolving power under conditions of pure energy spread has been known for more than 40 years. The method was published by W. C. Wiley and I. H. McLaren, xe2x80x9cTime-of-Flight Mass Spectrometer with Improved Resolutionxe2x80x9d, Rev. Scient. Instr. 26, 1150, 1955. The authors called the method xe2x80x9ctime-lag focusingxe2x80x9d (TLF). It has been applied to MALDI ionization quite recently under a variety of names, such as xe2x80x9cspace-velocity correlation focusingxe2x80x9d (xe2x80x9cSVCFxe2x80x9d: U.S. Pat. No. 5,510,613; Reilly, Colby and King) or xe2x80x9cdelayed extractionxe2x80x9d (xe2x80x9cDExe2x80x9d: U.S. Pat. No. 5,625,184; Vestal and Juhasz), and is also available in commercially available time-of-flight mass spectrometers.
The basic principle of this method is simple: the molecules and ions in the cloud are initially allowed to fly through a field-free region, without any electrical acceleration. This causes the faster molecules and ions to disperse further from the sample support electrode than do the slower ones, so that the speed distribution of the molecules and ions transforms to a spatial distribution. During this time, ionization by protons is also completed; those ions that are created from the molecules at a later time also demonstrate the same strict correlation of velocity and location. Only then is the acceleration of the ions switched on. The ions are accelerated by a homogenous acceleration field, with a linearly decreasing acceleration voltage. The faster ions are then more distant from the sample support electrode, which subjects them to a somewhat smaller acceleration voltage, and this gives them a rather lower final velocity for the drift region of the time-of-flight spectrometer than those ions that were initially travelling more slowly. If the time lag (or time delay) and the voltage drop (i.e. the strength of the accelerating field) are correctly chosen, then those ions that were initially slower, but which, following the acceleration, are travelling faster are able to catch up with those that were initially faster (but which, following acceleration, are travelling more slowly) at an adjustable location, the time-focus. This means that at this time-focus, the ions are dispersed with reference to mass, but those with the same mass are precisely focused in respect of the flight time.
Following removal of all the ions, the ion source potentials must be returned to the potentials required at the time of the next ionization process by the laser pulse.
In a linear time-of-flight mass spectrometer with no reflector, the time-focus is placed at the detector position through the selection of the delay time and the potential drop. In this way, a linear time-of-flight mass spectrometer achieves high mass resolution. Unfortunately, the time-focus depends slightly on the mass, so that the maximum resolution can only be achieved for one part of the spectrum, and is noticeably inferior in other parts of the spectrum.
A procedure has been published in patent DE 19638577 (Franzen) showing how it is possible to largely overcome the mass dependency of the time-focusing at the location of the detector in a linear mass spectrometer through modifying the accelerating field during time (pulse shaping), generating a good mass resolving power over the whole range of the mass spectrum. After the acceleration pulse has been switched on, the acceleration field is increased smoothly approaching a limit value. This procedure is referred to here as the procedure xe2x80x9cwith time-shaped acceleration pulsexe2x80x9d, or as xe2x80x9cpulse shape focusingxe2x80x9d.
In a time-of-flight mass spectrometer with a reflector, the time-focus of the acceleration is placed between the ion source and the reflector (U.S. Pat. No. 5,654,545; Holle, Kxc3x6ster and Franzen). The velocity-focusing reflector is then adjusted in such a way that ions of the same mass that leave this time-focus at the same time but with slightly differing velocities are again focused on the detector with reference to their velocities. The focus length of the reflector for ions of different velocities again depends slightly on the mass of the ions. Using the process of pulse shape focusing described above, it is again possible here to give the mass spectrum a uniform resolution over the entire range of masses. However, the intermediate time-focus, located between the ion source and the reflector, is not at the same position for ions of different masses.
The reflector of the time-of-flight mass spectrometer can also be used for the investigation of daughter ions (also known as fragment ions), created by metastable or collisionally induced decomposition of particularly selected ions. This selected type of ions is known as the xe2x80x9cparentxe2x80x9d or xe2x80x9cprecursorxe2x80x9d ion. Note: in the following text, mass spectra of ions that have not decomposed are referred to as xe2x80x9cprimary spectraxe2x80x9d, in contrast to the spectra of fragment or daughter ions. The primary spectra thus contain signals from all ions which can be used as the precursor ions, from which it is possible to generate daughter ion spectra.
In the MALDI ionization process, the ions in the vapor cloud generated by the laser pulse experience a large number of collisions, and this increases the internal energy of the ions by exciting internal oscillations. Depending on the energy density in the small focus area of the laser pulse, a greater or smaller number of these ions become xe2x80x9cmetastablexe2x80x9d, so that they decompose with a half-life in the order of a few microseconds; they decay when they are still in the first flight path of the mass spectrometer, which means that it is possible to detect the fragment ions in the mass spectrometer. Detection of fragment ions being thus generated in the mass spectrometer""s first field-free flight path by the reflector of a time-of-flight spectrometer is known as the PSD method (PSD=post source decay). It is, however, also possible to pass the precursor ions through a cell filled with collision gas, to cause collisionally induced decomposition (CID), and to detect the CID fragment ions in the same way.
If fragment ions are created by decomposition of ions following acceleration, then all the fragment ions continue to fly with the same velocity, v, as their precursor ions, although because of their lower mass, m, they have less kinetic energy, Ek=mv2/2. Due to their lower kinetic energy, they do not penetrate so far into the reflector""s second deceleration field; they therefore return earlier, and can be separately measured, according to their mass, at the end of the second field-free flight path.
However, a two-stage reflector can only ever measure a restricted portion of the full spectrum of daughter ions. For a gridless reflector with energy and space angle focusingxe2x80x94otherwise a very useful devicexe2x80x94it is therefore necessary to measure the daughter ion spectra in, for instance, a sequence of 14 spectrum segments, and then to piece the various segments together. This increases sample consumption and analysis time required to an unacceptable degree. A solution is offered in patent DE 19856014 (Holle, Kxc3x6ster and Franzen, U.S. Pat. No. 6,300,627), where the ions are subjected to post-acceleration through a sudden increase in the potential of the ions during their flight through a small potential cell (the daughter ion spectrum acquisition process with xe2x80x9cpotential liftxe2x80x9d).
In order not to superimpose the spectrum of the fragment ions of the desired parent ions by other xe2x80x9cparentxe2x80x9d ions and their decomposition products, it is necessary to deflect the undesired ions. For this purpose, an electrical deflection capacitor is used between the ion source and the reflector. A voltage applied to the capacitor plates generates a deflecting field, diverting the undesired ions and preventing them from reaching the ion detector. To permit passage of the desired ions the capacitor voltage is briefly removed, so that these ions can pass through undeflectedly. Once the ions have passed through, the voltage is switched on again, and further ions can no longer reach the detector. The mass resolution achieved by such a setup is in the region of R=60 to 80, which means that for ions with a mass in the region of 1,000 atomic mass units, the admission window is between 12 and 15 mass units wide.
The resolution can be greatly improved through bipolar switching, in which a positive deflection potential for the passage of the precursor ions that are to be selected is first switched to zero and then to a negative value. The resolution achievable in this way (in association with an appropriately designed capacitor) is around R=200 to 1,000, adequate for almost all applications. The unit supplying the deflecting field must therefore permit the deflecting field to be switched off within a very short period of time (a few nanoseconds) and then, after a predetermined interval (a few tens of nanoseconds) to be switched on again in the opposite direction. Between the spectrum acquisition processes, the voltage must be returned to the first polarity, so that each spectrum is acquired under the same conditions.
If the selector is to achieve high mass resolution, it is necessary for the time-focus of the delayed acceleration to be placed accurately within the selector. Because the location of the time-focus depends on mass, the parameters of the delayed acceleration (i.e. the delay period and, most importantly, the strength of the accelerating field) must be adjusted according to the mass of the ions that are to be selected, in order to achieve optimum resolution in the precursor ion selector. This is the second of the problems, mentioned briefly above, that still has to be solved.
The ion selector can select the ions in the first field-free flight path either before or after decomposition. As they decompose, the ions do not change velocity (at least not significantly), so that the precursor ions can be selected together with their daughter ions travelling at the same velocity.
The acquisition of daughter ion spectra is of particular significance in proteomics, in which the xe2x80x9cfingerprintxe2x80x9d spectra of peptide mixtures are initially acquired. The peptide mixtures are obtained through enzyme digestion of the protein under investigation. When required, and for confirmation, daughter ion spectra from selected digestion peptides may be measured. The digestion peptides from, for instance, tryptic digestion have lengths corresponding to between 500 and 4,000 atomic mass units.
As mentioned above, it is of great importance for the quality of the precursor ion selector that the focus of the delayed acceleration is located precisely within the precursor ion selector. Since, however, the method of delayed acceleration has a mass-dependent focus length, the parameters of the delayed acceleration, in other words the delay time and the accelerating field strength, must be adjusted in such a way that the time-focus for the ion mass to be selected (having, for instance, between 500 and 4,000 atomic mass units) is always located precisely within the precursor ion selector. This modification of the ion source potential and the switching time, however, again causes all the potentials to drift, and it is necessary to wait until equilibrium has once more been achieved. This makes a high sample throughput rate difficult.
It can thus easily be seen that a modern time-of-flight mass spectrometer has complicated electronics that must deliver and then reset a large number of synchronized voltage pulses, initially triggered by the laser. The ion source requires a mass-adjusted acceleration pulse (sometimes called ion extraction pulse) following a delay relative to the pulse of laser light, and a resetting of the voltages in addition to a continuously present main acceleration voltage. The ion selector needs bipolar switching and resetting under extremely precisely time control. The post-acceleration unit again uses precisely delayed voltage pulse switching, and a time-shaped acceleration pulse in addition to subsequent resetting. The requirements for precision in the switching times are extremely tight, and are of the order of fractions of a nanosecond. The requirements for reproducibility of the voltages are also extremely high; for critical voltages they are of the order of fractions of a volt. In the methods of operation used hitherto, there is a further difficulty created by the need to readjust the potentials of the ion source, depending on the masses of the precursor or ions, between one sample and the next.
It must further be possible to measure the flight times of the ions to within fractions of a nanosecond. This requirement places extremely high demands on the constancy of all the time delays, acceleration voltages and their pulse shapes. It is well known that thermal conditions of the voltage pulse supplies have effects both on the times and on the voltages. There are, however, also electrical effects in capacitors (resulting from the recovery of residual voltages) that disturb the reproducibility of electrical processes, if these are not repeated at precisely equal intervals.
The third problem area involves the homogenous and reproducible preparation of the samples for MALDI ionization. Modem procedures for MALDI time-of-flight mass spectrometry have accepted that samples will not be homogenous, and have attempted to solve the problems created in this way by reading every individual mass spectrum from the transient recorder, checking its quality, and only adding it to the sum spectrum if the quality is acceptably good. At the same time, the data from the poor quality spectra is fed back to assist control of the MALDI process. The feedback governs both the laser energy density and the selection of the point on the sample that is evaporated by the laser focus. The preparations are found to have xe2x80x9chot spotsxe2x80x9d that are particularly favorable for the spectrum measurements. The acquisition frequency for individual spectra, therefore, is limited to about 3 Hertz for spectrum transfer and evaluation. For future procedures with high sample throughput, this approach is of no use. Feedback may only be used in exceptional cases.
A solution to this problem is, however, in sight. In patent DE 197 54 978 (Schxc3xcrenberg and Franzen), a method of preparation has been published wherein special sample supports have hydrophilic anchors within a hydrophobic environment, achieving samples with precise localization and controlled shape and a fine, crystalline structure. In combination with automatic application of the sample droplets by a pipette robot, it is possible to achieve remarkably homogenous sample preparation. Recipes and formulas must be observed with extreme precision here. Preparing samples in this way provides a basis for the acqusition of spectra with high sample throughput.
The invention makes use of ionization by laser desorption, in particular by matrix assisted laser desorption and ionization (MALDI), with improved resolution by delayed ion acceleration. There is a generation of daughter ions through decomposition after leaving the ion source (post source decay: PSD) or by impact fragmentation (collisionally induced decomposition: CID). The precursor and daughter ions are selected by a precursor ion selector, and post-acceleration of the ions before they reach the reflector may be employed.
A first basic idea of the invention is to constantly run the periodic sequence of voltage pulses on the acceleration electrodes in the ion source under the control of a clock generator running at a fixed basic frequency, irrespective of whether a spectrum is being acquired or not. For lasers which are switched off during non-acquiring pauses, this is done in such a way that if no spectrum is being acquired the clock generator will trigger the sequence of voltage pulses directly, but if a spectrum is to be acquired, it will trigger the laser, whose light pulse in turn triggers the voltage pulses. In this way it is possible to bridge pauses, without loss of thermic and electic equilibrium, and with no unnessessary diminishing of laser life time. Such pauses might arise from movement of the sample support when it is necessary to bring a new sample into the laser""s focus location. It has been found that the tiny irregular phase differences of a few tens of nanoseconds, generated by the jitter of the laser, and even longer delays of a few microseconds, necessary to select the parent ions in the parent ion selector, can be neglected, because they do not affect the established equilibrium.
It is possible, for instance, for the clock generator to be set to a frequency that corresponds to the fastest pulse frequency of which the laser is capable, but it can also be set to a multiple of that frequency. For most MALDI procedures on temperature-sensitive samples, about 20 Hertz represents the upper limit for the pulse rate, as the samples will otherwise become overheated. The procedure could, however, also be used at significantly higher pulse rates, if these can be usefully applied.
The invention also makes it possible to decouple the frequency with which spectra are acquired from the basic frequency, when a spectrum is not acquired in every electrical period. If the base frequency, for instance, is 20 Hertz, then the frequency with which spectra are acquired can, for instance, be reduced to 10 or 5 Hertz, by using only every second or fourth period of the base frequency, should the sample or the measurement process require such a procedure. If the base frequency represents a multiple of the fastest laser pulse frequency, then intermediate levels that do not immediately equate to a reduction by a factor of two may also be set. It is also possible for electrical periods to be selected on an irregular basis for the acquisition of spectra. Individual or multiple periods can be omitted, should this be required for the purposes of fetching spectra from the transient recorder or the calculation of feedback adjustments.
Adjustments to the voltage during the acquisition of primary spectra, disturbing the equilibrium, can be avoided by pulse-shape focusing in accordance with DE 19638577. The time-focusing of all ion masses exactly at the location of the detector is achieved here, and this brings a uniformly good mass resolving power over the entire mass spectrum. This means that any adjustment of voltages in the ion source according to the particular samples can be avoided. The pulse-shape focusing process is therefore an essential precondition for high spectral throughput. It has been found that, in many cases, a relatively simple exponential function is sufficient (in particular when a central electrode is used, as in FIG. 1), in which the voltage of the acceleration pulse approaches a limit voltage exponentially. This kind of voltage curve can be achieved with a simple R-C network.
For the sake of a high throughput of spectral measurements it is also helpful to first measure the primary spectra of a large number of samples, preferably all the samples, on one sample plate, then to make the adjustments for the measurements of the daughter ion spectra, and then, if necessary for the purposes of analysis, to measure the daughter ion spectra of the samples. In order to be able to carry out this process, the primary spectra are passed immediately after being acquired (in real time, so to speak) to an expert system. This system determines the necessity of obtaining daughter ion spectra, and calculates the associated precursor ion masses to be used for the daughter ion spectra. The necessity is defined here in accordance with the analytic task.
The daughter ion spectra are measured by reducing the acceleration voltage in the ion source and introducing a pre-cursor ion selector and a post-acceleration unit, both of which are mounted between the ion source and the reflector. They can be removed from the path of the ions to acquire the primary spectra.
There remains, for the measurement of daughter ion spectra, the still unsolved problem of avoiding the adjustment of the ion source potentials from one daughter ion spectrum to the next. These are made necessary, because the daughter ions to be measured are derived from precursor ions that are different in each case, and the time-focus of each must be placed within the precursor ion selector.
It is therefore a further basic idea of the invention to make the location of the time-focus in the precursor ion selector independent of mass with the aid of a new mode of operation for the xe2x80x9cpulse-shape focusingxe2x80x9d, which until now has always been aimed at achieving an even resolution across the entire spectrum. This means that the sequence of delayed, time-shaped acceleration pulses in the ion source can also be made to run in exactly the same way for the daughter ion spectra from one spectrum to another, without additional adjustment, independently of the mass of the precursor ion that is to be selected. This requires the acceleration pulse to have a voltage, rising with time, of a form to be determined experimentally, so that the focus position in the precursor ion selector becomes independent of mass. It has also been found here that a relatively simple exponential function is sufficient, in which the voltage of the acceleration pulse exponentially approaches a limit value. This voltage curve can also be achieved with a simple R-C network, whose time constant, however, is different from that required for the acquisition of primary spectra. Between acquisition the primary and the daughter ion spectra, it is therefore necessary to switch between the R-C networks.
The slight dependency of the reflector""s focus length on the mass of the ions can be compensated for through an appropriately time-shaped acceleration pulse in the post-acceleration unit, as is basically described in patent application DE 100 34 074.1. Once again, this permits the voltage curves to be continuously repeated, without needing to make adjustments between spectrum acquisitions.
The shaped voltage pulses in the precursor ion selector and in the post-acceleration unit (the potential lift) are also allowed to run at the base frequency. For the selection of the masses, the flight time of the ions from the ion source to the precursor ion selector is the significant factor, thus the delay of the selector""s passing window with reference to the start time of the ions from the ion source. This variation in the delay of the passing period, in comparison with the start time, is a small but precise phase shift in the sequence of the voltage pulses, and this can be implemented without significantly disturbing the electronic equilibrium. The phase shift is a matter of only a few microseconds compared with a basic clock generator cycle of, for instance, 50 milliseconds; it must, however, be possible to adjust the phase shift with nanosecond precision.
Similar considerations apply to the post-acceleration unit, with its rather complicated voltage pulse scheme; here again, only a small but precise phase shift is applied.
The reflector always remains at a constant potential.
It is not essential for the fragmentation of the precursor ions to be initiated by creating metastable ions in the MALDI process itself. It is also possible to fit a collision chamber filled with collision gas between the ion source and the precursor ion selector, or between the precursor ion selector and the post-acceleration unit. This will create daughter ions through impact fragmentation (CID=collisionally induced decomposition).